As cell cycle regulators whose activity is frequently alteredin human
cancers, cyclin-dependent kinases (cdks) are noveltargets for
therapeutic intervention. cdk inhibition is an emergingstrategy for
the treatment of non-small cell lung carcinomas(NSCLCs) because most
derived cell lines express functionalretinoblastoma protein (Rb) but
appear to bypass its functionwith inappropriate cdk activity. Elevated
cdk4/cdk6 activityin NSCLC cells is often due to inactivation of the
p16Ink4acdk inhibitor. To model the effects of cdk4/cdk6
inhibition,we have expressed p16Ink4a in a Rb-positive
NSCLC cell linethat lacks endogenous p16Ink4a expression.
Whereas cdk4/cdk6inhibition and Rb dephosphorylation are expected on
p16Ink4aexpression, we have also observed indirect cdk2
inhibition.cdk2 inactivation by the redistribution of other cdk
inhibitorsmay be required for p16Ink4a-mediated growth
suppression ofRb-positive cells. The implications of such a
requirement onthe use of chemical cdk inhibitors to treat human
cancers willbe discussed.

Although Rb is phosphorylated in vivo by several cyclin-cdk
complexes,including D-type cyclins (cyclins D1, D2, and D3) bound to
cdk4or cdk6, cyclin E bound to cdk2, and cyclin A bound to eithercdk2
or cdc2, its initial phosphorylation and inactivation in
G1phase is a point of controversy. Rb contains
at least 16 consensuscdk phosphorylation sites; although the
functional significanceof multiple, sequential phosphorylations is not
completely understood,several phosphorylation sites are known targets
of cyclin D-cdk4/cdk6and cyclin E-cdk2 kinases (29, 30)
.
Initial Rb phosphorylationby cyclin D-cdk4/cdk6 appears to enable
cyclin E-cdk2 activation,leading to further Rb phosphorylation and E2F
release (24, 31,32, 33)
.

G1 phase cdks can be regulated by the binding of
inhibitoryproteins of two structurally distinct classes. Both the Ink4
(p16Ink4a,p15Ink4b,
p18Ink4c, and p19Ink4d) and
Cip/Kip (p21Cip1/Waf1,
p27Kip1,and p57Kip2)
families of proteins can inhibit cdks, but in responseto different
antiproliferative stimuli and with different specificitiesand
mechanisms (3, 34)
. The Ink4 proteins are characterizedby
ankyrin-like repeats and by their exclusive binding to cdk4and cdk6
(35)
. Cip/Kip proteins share a homologous
NH2-terminaldomain with both cyclin and
cdk-binding regions, and bind awide range of cyclin-cdk complexes
including cyclin D-cdk4/cdk6,cyclin E-cdk2, and cyclin A-cdk2
(36)
. Individual Ink4 andCip/Kip proteins can be
permanently induced during differentiationor senescence or transiently
expressed in response to antiproliferativesignals (34, 37, 38)
. p16Ink4a and possibly
p15Ink4b aretumor supressors inactivated in
various human cancers, whereasother cdk inhibitors do not appear to be
targeted for inactivationat such a high frequency (39)
.
Although no inactivating mutationshave been detected in any Cip/Kip
protein, reduced or absentp27Kip1 expression has
been reported in various human cancersand is correlated with a poor
patient prognosis (40, 41, 42,43)
.

Because most normal cells are quiescent and lack cdk activity,chemical
cdk inhibition may slow the proliferation of tumorcells with minimal
effects on normal tissue. cdk inhibitionas a therapy for lung cancer,
frequently in combination withgenotoxic agents such as cisplatin and
Taxol (46, 47, 48)
,is currently under clinical evaluation.
Chemical cdk inhibitorscurrently available include the selective
compounds olomoucine,butyrolactone, and flavopiridol and the
nonselective inhibitors7-hydroxystaurosporine (UCN-01) and suramin
(49, 50, 51)
. Cellcycle studies using these compounds in
various mammalian cellculture models have confirmed their effect on
cell cycle progressionat both the G1-S- and
G2-M-phase transitions (49, 50, 52)
.
cdkinhibitors can also either induce or inhibit apoptosis, depending
onwhether cells are actively proliferating or synchronized tospecific
cell cycle positions with chemotherapeutics (46, 48, 49, 53, 54)
.

The identification of p16Ink4a as a tumor
suppressor inactivatedin various human malignancies has prompted a
search for specificcdk4/cdk6 chemical inhibitors. Although no
cdk4/cdk6-selectivecompounds have been described to date,
high-throughput screeningand structural refinement will likely lead to
their development(55, 56, 57)
. Currently, the only available
cdk4/cdk6 inhibitoris flavopiridol, which is highly specific for cdks
but is notselective for the cdk4/cdk6 subfamily (51, 58)
.
Because theloss of p16Ink4a expression in NSCLC
is important for maintenanceof the transformed phenotype,
cdk4/cdk6-specific inhibitorsmay be of use in the treatment of
Rb-positive lung cancers.To model the biochemical effects of cdk4/cdk6
inhibition bya selective cdk inhibitor, we have overexpressed the
cdk4/cdk6-specificinhibitor p16Ink4a in a
Rb-positive NSCLC cell line that lacksendogenous
p16Ink4a protein expression (NCI-H460).

Our results demonstrate that in addition to inhibiting cdk4and cdk6,
p16Ink4a expression in NCI-H460 cells indirectly
inhibitscdk2. Whereas cdk4 and cdk6 inhibition can be attributed to
directp16Ink4a binding, cdk2 inactivation is
mediated by the redistributionof p21Cip1/Waf1
and p27Kip1 proteins from cyclin D-cdk4/cdk6
complexesto cyclin E-cdk2 complexes. cdk2 inhibition may be required
forp16Ink4a-mediated cell cycle arrest of
Rb-positive NSCLC cells,as has been shown for Rb-positive human
osteosarcoma (U2OS)cells (59)
. If small molecule cdk
inhibitors do not functionsimilarly to p16Ink4a
in displacing p21Cip1/Waf1 and
p27Kip1,cdk4/cdk6 inhibition by chemotherapy
will not be equivalentto cdk4/cdk6 inhibition by gene-based therapy
and may thereforeprove a less effective therapy.

Results

Cyclin Expression and Rb Phosphorylation in NCI-H460 Cells Is
Consistent with a Normal Cell Cycle.
Before introducing a cell cycle regulator such as
p16Ink4a tocells lacking endogenous expression,
we wished to characterizetheir pattern of cyclin expression and Rb
phosphorylation. Isolationof synchronous populations of cells
demonstrated that despitetheir rapid proliferation and transformed
phenotype, NCI-H460NSCLC cells have an apparently normal
profile of cyclin expressionand Rb phosphorylation. FACS profiles of
cells enriched forearly G1 phase, late
G1 phase, S phase, and G2-M
phase are presentedin Fig. 1A
. In Fig. 1B
, increasing elutriator fraction
numberscorrespond to samples of increasing cell size and cell cycle
transit,beginning with the smallest, early G1
phase cells. Beginningwith G1 phase cells,
cyclin D1 is the first cyclin whose expressionreaches a maximum level
before declining, followed closely bycyclin E as cells approach the
G1-S-phase boundary, then bycyclin A as cells
transit S phase, and finally by cyclin B1as cells enter mitosis (Fig. 1B)
. Although its levels do notchange appreciably in
synchronous cells of some lineages, cyclinD1 protein cycles in
proliferating NCI-H460 cells. The patternof Rb phosphorylation is also
characteristic of a normal cellcycle because early
G1 phase cells have Rb in a faster-migrating,
hypophosphorylatedform, which gradually shifts to a more slowly
migrating, phosphorylatedform as cells reach the
G1-S-phase transition (Fig. 1B)
. Rb
levelsin fractions 28 of Fig. 1B
appear to be reduced due
tosmearing of partially phosphorylated forms. At this level of
analysis,the rapid proliferation of NCI-H460 cells is consistent with
thederegulation of cell cycle entry, and not the cell cycle machinery
itself.

Fig. 2. A, effect of ectopic p16Ink4a expression on
the proliferation of NCI-H460 cells, which lack endogenous
p16Ink4a protein. Exponentially growing cells were
collected, infected at a MOI of 100 with recombinant adenoviruses
encoding either GFP (Ad-GFP; ) or p16Ink4a
(Ad-p16Ink4a; ), plated in triplicate at an initial
density of 4 x 105 cells/6-cm dish, and incubated. At
the indicated times, cells were harvested and counted using a Coulter
Multisizer (Coulter Electronics Inc., Hialeah, FL). Proliferation
assays were limited to 48 h because adenovirus genomes are not
stably maintained. B, effect of p16Ink4a
expression on the cell cycle distribution of NCI-H460 cells, as
measured by FACS analysis. Asynchronously proliferating cultures were
infected with Ad-p16Ink4a or a control virus (Ad-GFP) as
described in A and harvested for FACS analysis after
24 h of incubation. Whereas GFP expression had no observable
effect on cell cycle distribution, p16Ink4a expression
resulted in an accumulation of cells in G0-G1
phase, seen as an increase in the left-most peak, which
widens due to cell growth without division. C,
expression of cell cycle regulators in uninfected, control infected
(Ad-GFP), and Ad-p16Ink4a-infected NCI-H460 cells. Log
phase cultures were infected at the indicated MOIs and harvested after
24 h of incubation. Protein extracts were prepared and analyzed by
Western blotting with antibodies to various cell cycle regulatory
proteins, using actin as a loading control.

Whereas GFP expression had no discernable effect on cell proliferation,
cellsinfected with Ad-p16Ink4a at MOIs of 10 and
100 showed an enrichmentof cells with a
G0-G1 content of DNA and a
cyclin expressionconsistent with a G1-phase cell
cycle arrest (Fig. 2, B andC)
. The
G0-G1 peak in
p16Ink4a- expressing cells is wider dueto
some continued cellular growth without division. Arrestedcells showed
expression of cyclins D1 and E and an absence ofcyclin A and B1
proteins expressed during S phase and M phase,respectively (Fig. 1B)
. Analysis of protein extracts preparedfrom
p16Ink4a-arrested cells also identified Rb in a
predominantlyhypophosphorylated form, consistent with
G1 cdk inhibition andcellular
G1-phase arrest. Detection of cyclin E protein
duringa p16Ink4a-mediated arrest was not
expected because its expressionis largely regulated by E2F, and Rb
hypophosphorylation (Fig.2C)
represses E2F activity.
Previous studies involving induciblep16Ink4a
expression in osteosarcoma cells have also reportedsustained cyclin E
protein levels during p16Ink4a expression
(59, 63), and we have observed the same pattern of
cyclin expressionin primary lung epithelial (NHBE) cells after
Ad-p16Ink4a infection.4
CyclinE transcription and protein stability during
p16Ink4a expressionare currently under
evaluation because E2F-independent mechanismsmay contribute to the
regulation of cyclin E levels.

No cellular apoptosis was apparent during
p16Ink4a expressionbecause cell numbers did not
decrease during a proliferationassay, and no cells with a subdiploid
DNA content were detectedby FACS analysis (Fig. 2, A and B)
. Importantly, p16Ink4a expression
didnot increase the levels of the cdk inhibitors
p21Cip1/Waf1 andp27Kip1
during suppression of NCI-H460 cell growth (Fig. 2C)
.

Ectopically Expressed p16Ink4a Inhibits cdk4, cdk6, and
cdk2 without Binding cdk2.
Because ectopic p16Ink4a expression in NSCLC
cells induced acell cycle arrest with reduced Rb phosphorylation, we
measuredthe in vitro activity of cdk4, cdk6, and cdk2
before and afterp16Ink4a introduction (Fig. 3A)
. As expected, cdk4, cdk6, andcyclin D1- and D3-associated
kinase activity on GST-Rb was reducedon p16Ink4a
expression (cyclin D2 protein expression was notdetectable in NCI-H460
cells). Additionally, in the absenceof induction of the cdk2
inhibitors p21Cip1/Waf1 and
p27Kip1,cdk2 activity was eliminated after
p16Ink4a introduction. Thecomplexity of cdk
regulation offers several possible mechanismsby which cdk2 can be
inhibited. Coimmunoprecipitation experimentsdemonstrated that whereas
direct cdk binding can account forthe effects of
p16Ink4a on the activity of cdk4 and cdk6, it
cannotexplain the associated cdk2 inhibition because no binding was
detectable(Fig. 3B)
. The lack of cdk2 binding is consistent
with thespecificity of p16Ink4a because no
association of p16Ink4a witheither cdk2 or cdc2
in any cell type has been reported in theliterature.

Fig. 3. A, effect of p16Ink4a expression on the
in vitro kinase activity of cdk4, cdk6, cyclin D1,
cyclin D3, and cdk2 immunoprecipitates. The indicated proteins were
immunoprecipitated from uninfected, control-infected (Ad-GFP), and
Ad-p16Ink4a-infected NCI-H460 cell extracts and used in an
in vitro kinase assay with [-32P]ATP
and GST-Rb as substrate. B, coimmunoprecipitation of
p16Ink4a with cdk4, cdk6, and cdk2 from uninfected,
control-infected (Ad-GFP), and Ad-p16Ink4a-infected
NCI-H460 cell extracts. cdk4, cdk6, and cdk2 immunoprecipitates were
boiled in SDS-PAGE sample buffer, resolved by gel electrophoresis, and
Western blotted with antibodies to the immunoprecipitated protein and
p16Ink4a.

p16Ink4a Expression Redistributes
p21Cip1/Waf1 and p27Kip1 from cdk4/cdk6 to
cdk2.
Whereas ectopic p16Ink4a expression in NSCLC
cells did not affectthe levels of p21Cip1/Waf1
or p27Kip1, the ability of Cip/Kipproteins to
bind various cyclin-cdk complexes allows for theirredistribution. Ink4
and Kip proteins are known to collaboratein arresting the growth of
mink lung epithelial (Mv1Lu) cellsexposed to the inhibitory cytokine
TGF-ß (64)
. In thiscase, TGF-ß induces
p15Ink4b expression, promoting theformation of
inactive p15Ink4b-cdk4 and
p15Ink4b-cdk6 assemblies.Before TGF-ß
treatment, p27Kip1 is found in cdk2, cdk4,and
cdk6 complexes. Because Ink4 and Cip/Kip proteins shareoverlapping
binding sites on cdk4 and cdk6, p15Ink4b
inductionredistributes p27Kip1 from cdk4 and
cdk6 complexes to cdk2 complexes(64, 65)
.
Immunoprecipitation of cdk complexes before andafter ectopic
p16Ink4a expression demonstrated that Ink4
proteinexpression in NCI-H460 cells similarly redistributes Cip/Kip
proteinsfrom cdk4/cdk6 complexes to cdk2 complexes (Fig. 4A)
. Controlvirus-infected cells show
p21Cip1/Waf1 and p27Kip1
associatedwith cdk2, cdk4, and cdk6 complexes. When ectopically
expressed,p16Ink4a binds cdk4 and cdk6 at the
expense of p21Cip1/Waf1and
p27Kip1. Without an observable change in the
absolute Cip/Kipprotein levels in the cell (Fig. 2C)
, the
amount of p21Cip1/Waf1and
p27Kip1 bound to cdk2 increases after
p16Ink4a expression,at a time point when kinase
assays indicate that all cdks areinhibited (Figs. 3A
and 4A)
.

Fig. 4. A, effect of p16Ink4a expression on the
levels of p21Cip1 and p27Kip1 associated with
cdk2, cdk4, and cdk6 in NCI-H460 cells. cdk2, cdk4, and cdk6
immunoprecipitates from control-infected (Ad-GFP) and
Ad-p16Ink4a-infected cells were boiled in SDS-PAGE sample
buffer, resolved by gel electrophoresis, and Western blotted with
antibodies to the immunoprecipitated protein, p21Cip1 and
p27Kip1. For cdk2, coimmunoprecipitation of cyclin E was
also assayed. B, identical coimmunoprecipitation
experiments performed with lysates prepared from control-infected and
Ad-p16Ink4a-infected NCI-H661 cells. C,
identical coimmunoprecipitation experiments performed with lysates
prepared from control-infected and Ad-p16Ink4a-infected
primary lung epithelial (NHBE) cells.

Although p21Cip1/Waf1 and
p27Kip1 redistribution to cdk2 complexesmay be
sufficient for cdk2 inhibition, other mechanisms mayalso contribute.
Cyclin absence may explain the lack of cdk2activity; however,
coimmunoprecipitation demonstrates that cyclinE remains associated
with cdk2 and even appears to be up-regulatedduring
p16Ink4a expression (Fig. 4A)
. We have
not investigatedp57Kip2 induction or cdk2
phosphorylation status, and we cannotexclude their involvement in the
observed inhibition of cdk2activity.

To determine whether Cip/Kip redistribution is unique to NCI-H460
cells,we performed similar coimmunoprecipitation experiments witha
second Rb-positive NSCLC cell line (NCI-H661), and NHBE cells.Although
cdk2 activity was not assayed, in both cell types adenovirus-mediated
p16Ink4aexpression led to
G1-phase cell cycle arrest (data not shown),a
decrease in p21Cip1/Waf1 and
p27Kip1 association with cdk4and cdk6, and a
simultaneous increase in association with cdk2.In control
virus-infected cells, Cip/Kip proteins are boundto cdk2, cdk4, and
cdk6; p16Ink4a expression disrupts
p21Cip1/Waf1and p27Kip1
interactions with cdk4 and cdk6, promoting theirrelocation to cdk2
(Fig. 4, B and C)
. As was observed for NCI-H460
cells,p16Ink4a expression had no effect on the
total levels of p21Cip1/Waf1and
p27Kip1 protein (data not shown), and cyclin E
remainedassociated with cdk2 during Cip/Kip protein redistribution
(Fig.4, B and C)
.

Overexpression of p16Ink4a and induction of
p15Ink4b thereforeappear to be equivalent in
displacing Cip/Kip proteins fromcyclin D-cdk4/cdk6 complexes, leading
to the increased associationof p21Cip1/Waf1 and
p27Kip1 with cyclin E-cdk2 complexes andthe
subsequent inhibition of cdk2 kinase activity, as shownin the present
study. Physiological induction of p16Ink4a in
primarycells can occur during replicative senescence or in responseto
constitutive mitogenic signaling as part of a proposed tumor
surveillancemechanism (66, 67, 68, 69)
. Consistent with these
findings,primary lung epithelial (NHBE) cells show elevated
p16Ink4aprotein after extended cultivation or
during ectopic expressionof activated H-Ras and are sensitive to
adenovirus-mediatedp16Ink4a
overexpression.4 Our demonstration of
p21Cip1/Waf1and p27Kip1
redistribution during p16Ink4a expression in
primary(NHBE) cells argues that this response to Ink4 protein
accumulationis not limited to established cell lines. Whether
physiologicalp16Ink4a accumulation, as seen
during primary cell senescence,has the same effect on Cip/Kip protein
interactions with cdksis under investigation.

Ink4 protein induction and Cip/Kip protein relocation to cdk2are
proposed to be a general mechanism of cdk inhibition andmammalian cell
growth arrest in response to various antiproliferativesignals
(70)
. Controversially, this model of inhibitor
redistributionproposes that p21Cip1/Waf1 and
p27Kip1 exist in active cyclinD-cdk4/cdk6
complexes before the elevation of Ink4 protein levels.Whether Cip/Kip
proteins are universal cdk inhibitors is anunresolved question, with
different experimental systems producingconflicting results
(71, 72, 73)
. The isolation of cyclin-cdkcomplexes with
kinase activity despite the presence of stoichiometricamounts of bound
p21Cip1/Waf1 and p27Kip1 is
difficult to reconcilewith available structural information for a
crystallized complexof cyclin A-cdk2-p27Kip1(74)
. In that trimeric structure,extensive interactions
between p27Kip1 and cyclin A-cdk2 dramatically
distortthe enzymes catalytic cleft and prevent ATP binding.Until
the structure of a cyclin D-cdk4/cdk6-Cip/Kip proteincomplex is
solved, it will be unclear how p21Cip1/Waf1 or
p27Kip1can exist in an active cyclin-cdk
complex, although a growingbody of evidence supports this (72, 75, 76, 77)
. Evolutionarydistance between cdk subfamilies may
explain differing sensitivitiesto inhibitory proteins and drugs. For
example, despite theirsimilarity to other cdks, cdk2 does not bind
Ink4 proteins,and cdc2 (cdk1) does not bind either Ink4 or Cip/Kip
proteins.

A recent phenotypic analysis of p21Cip1/Waf1 and
p27Kip1 doublynull mice indicates that the role
of Cip/Kip proteins is notlimited to cdk inhibition. Despite their
ability to inhibitcdk2 complexes, Cip/Kip proteins are essential for
the assemblyand nuclear localization of cyclin D-cdk4/cdk6 complexes
(77)
.Inhibitor redistribution and the identification of
p21Cip1/Waf1and p27Kip1 as
assembly factors suggest that Cip/Kip proteinsand cyclin D-cdk4/cdk6
kinases are functionally interdependent.Whereas cyclin D-cdk4/cdk6
complexes require p21Cip1/Waf1 and
p27Kip1for assembly, in the absence of Ink4
protein expression, thesecomplexes can act as reservoirs for Cip/Kip
proteins, limitingtheir ability to inhibit cyclin E-cdk2 enzymes. This
noncatalyticrole for cdk4 and cdk6 complexes is important to the
regulationof a normal cell cycle because mitogen-stimulated D-type
cyclinexpression in early G1 phase (Fig. 1)
leads to assembly ofcyclin D-cdk4/cdk6 complexes, sequestering Cip/Kip
proteinsand allowing the activation of cdk2 as cells approach S phase
(78, 79).

The observation of p16Ink4a inactivation in
various human cancershas prompted efforts to develop drugs that can
mimic its cellularfunction. However, compounds currently under
development asspecific cdk4/cdk6 inhibitors may not fully substitute
for p16Ink4aprotein during suppression of cell
growth. If cdk2 inhibitionis required for efficient cell cycle arrest,
chemical inhibitorsspecific to cdk4/cdk6 may only have a lasting
effect on cellgrowth if they redistribute Cip/Kip proteins to cdk2.
Whereassmall molecule inhibitors may effectively inhibit cdk4/cdk6
activity,they may be too small to displace associated Cip/Kip
proteins.If so, cdk4/cdk6 inhibition by chemotherapy will not be
mechanisticallyequivalent to cdk4/cdk6 inhibition by a gene-based
therapy.Chemical inhibition of cdk4/cdk6 may be more similar to
expressionof a dominant-negative form of cdk4 or a phosphorylation
site-deficientform of Rb, both of which do not affect cdk2 activity
and canonly temporarily arrest cell growth. When specific cdk4/cdk6
chemicalinhibitors are developed, an important functional test in
culturedcells will be whether p21Cip1/Waf1 and
p27Kip1 are redistributed,and whether cdk2
activity is inhibited.

Materials and Methods

Cell Culture.
NCI-H460 and NCI-H661 cells were originally established at theNational
Cancer Institute, [Bethesda, MD (83)
] and obtainedfrom
the University of Colorado Cancer Center Tissue CultureCore Facility.
Cells were cultured in RPMI 1640 with 10% fetalbovine serum in a
humidified 37°C incubator. NHBE cellsand serum-free bronchial
epithelial cell growth medium werepurchased from Clonetics. NHBE cells
were cultured in a humidified37°C incubator and expanded by
approximately six populationdoublings prior to use.

Immunoprecipitations were performed with 500 µg of lysateand 0.5
µg of antibody bound to 10 µl of proteinA- or protein G-Sepharose
(Amersham Pharmacia Biotech) by rotatingfor 3 h at 4°C. The
antibodies used to immunoprecipitatecdk2, cdk4, and cdk6 are the same
as those used for Westernblotting; antibodies to cyclins D1 and D3
were from NeoMarkers(clones DCS-11 and DCS-28.1). Immune complexes
were washed threetimes with cold HEPES/Tween lysis buffer and twice
with coldkinase buffer [50 mM HEPES (pH 7.5), 10
mM MgCl2, 2.5 mM EGTA,1
mM DTT, 10 mM ß-glycerophosphate, 1
mM NaF, 0.1 mM sodiumorthovanadate, and 20
µM ATP]. Samples were resuspendedin 30 µl of kinase
buffer containing 2 µg of GST-Rbfusion protein (see below) and 10
µCi of [-32P]ATP (AmershamPharmacia
Biotech); after incubation at 30°C for 30 minwith occasional mixing,
reactions were stopped by adding 30µl of hot 2x Laemmli sample
buffer and boiling for 5 min.Samples were resolved by SDS-PAGE in a
10% gel, and phosphorylatedproteins were detected by autoradiography.

GST-Rb fusion protein was prepared by growth and induction of
Escherichiacoli transformed with pGEX-Rb (amino acids
792928; Ref.87
). Cultures (100 ml) were grown to an
A600 nm of 0.6, andGST-Rb
expression was induced by the addition of 0.1 mM
isopropylthioglycosidefor 4 h. Cell pellets were resuspended in a
0.1 volume of coldNETN buffer, and cells were lysed by sonication on
ice. Glutathione-Sepharose4B (Amersham Pharmacia Biotech) was mixed
with cleared lysatesfor 4 h at 4°C and then washed three times
with NETN bufferand washed twice with kinase buffer. GST-Rb fusion
proteinswere eluted from glutathione-Sepharose beads by incubation in
kinasebuffer with 2 mM reduced glutathione
(Sigma) at 4°C. Theconcentration of soluble GST-Rb was estimated by
comparisonto known amounts of protein standards after SDS-PAGE and
CoomassieBlue staining.

Immunoprecipitations were performed with 1.5 mg of lysate and3 µg of
antibody bound to 30 µl of protein A- orprotein G-Sepharose by
rotating for 3 h at 4°C. The antibodiesused for
immunoprecipitation/kinase assays were also used for
immunoprecipitation/Westernblotting assays. Immune complexes were
washed three times withcold NETN buffer and boiled for 5 min in
Laemmli sample buffer.Samples were resolved by SDS-PAGE in 10% or
13% gels and Westernblotted for immunoprecipitated and
coimmunoprecipitated proteins.

Acknowledgments

We thank Maria C. Todd for critical reading of the manuscript
andJames V. DeGregori for assistance with adenovirus productionand
infections.

Footnotes

The costs of publication of this article were defrayed in partby the payment of page charges. This article must thereforebe hereby marked advertisement in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.